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2Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD,USA3Department of Medicine, Vanderbilt University, School of Medicine, Nashville,TN, USA4Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China5Department of Public Health Analysis, School of Community Health andPolicy, Morgan State University, Baltimore, MD, USA6Department of Epidemiology, The University of Texas MD Anderson CancerCenter, Houston, TX, USAAbstractBackgroundAssociations between polycyclic aromatic hydrocarbons (PAHs) and colorectal cancer havebeen reported previously but few studies have characterized PAH exposure using biologicalmeasurements. We evaluated colorectal cancer risk in relation to urinary concentration of 1-hydroxypyrene glucuronide (1-OHPG), a polycyclic aromatic hydrocarbon (PAH) metabolite,and assessed determinants of PAH exposure among controls in the Shanghai Women’sHealth Study (SWHS).MethodsConcentrations of 1-OHPG were measured in spot urine samples collected from 343colorectal cancer cases and 343 individually matched controls. Questionnaires wereadministered to collect information on demographic characteristics and reported exposures.Odds ratios were calculated for risk of colorectal cancer in relation to quartiles of urinary 1-OHPG concentration. Potential determinants of natural log-transformed urinary 1-OHPGconcentration were evaluated among a combined sample of controls from this study andanother nested case–control study in the SWHS (Ntotal=652).ResultsNo statistically significant differences in risk of colorectal cancer by urinary 1-OHPG levelswere observed. Among controls, the median (interquartile range) urinary 1-OHPGconcentration was 2.01 pmol/mL (0.95-4.09). Active and passive smoking, using coal as acooking fuel, eating foods that were cooked well done, and recent consumption of frieddough (e.g., yóutiáo) were associated with elevated levels of 1-OHPG, though only activesmoking and fried dough consumption achieved statistical significance in multivariateanalyses.

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ConclusionsThis study does not provide evidence of an association between urinary levels of 1-OHPGand risk of colorectal cancer among women. Several environmental and dietary sources ofPAH exposure were identified. Overall, the levels of 1-OHPG in this population ofpredominantly non-smoking women were considerably higher than levels typically observedamong non-smokers in Europe, North America, and other developed regions.Keywords1-hydroxypyrene glucuronide, polycyclic aromatic hydrocarbons, colorectal cancer, ChinaBackgroundPolycyclic aromatic hydrocarbons (PAHs) are byproducts of incomplete combustion oforganic materials. Individuals may be exposed to PAHs through various environmentalsources including tobacco smoke, ambient air pollution, and consumption of grilled foods [1].High levels of PAH exposure have been observed for various occupational activitiesincluding coal gasification, coke production, aluminum smelting, coal-tar distillation, andpaving and roofing with coal-tar pitch [2]. The International Agency for Research on Cancerhas classified PAH exposure in these occupational settings as a Group 1 carcinogen(“carcinogenic to humans”).Colorectal cancer is the third most common type of incident cancer in the world among bothmen and women [3]. There is some evidence of an increased risk of colorectal cancer inrelation to PAH exposure. Colorectal cancer mortality was elevated among PAH-exposed gasfurnace workers in an occupational cohort study in Germany [4], and several case–controlstudies have reported associations between estimated dietary intake of benzo(a)pyrene, aparticular PAH compound, and risk of colorectal adenoma [5-8]. In a prospectiveinvestigation within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial,higher estimated dietary benzo(a)pyrene intake was associated with an increased risk ofincident rectal adenoma but not colon adenoma [9]. Furthermore, active cigarette smoking, amajor source of PAH exposure, has also been consistently associated with an increased riskof colorectal cancer [10].However, few studies have evaluated the relationship between PAH exposure biomarkers andrisk of colorectal adenoma [11] or colorectal cancer [12]. In this nested case–control study,we evaluated the risk of colorectal cancer in relation to urinary concentration of 1-hydroxypyrene glucuronide (1-OHPG), a metabolite of pyrene and an established biomarkerof PAH exposure [13], among participants in the Shanghai Women’s Health Study. As asecondary analysis, we evaluated the relations between urinary 1-OHPG concentration andpotential determinants of PAH exposure among a combined sample of controls from thisstudy and a related nested case–control study of gastric cancer.

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MethodsStudy participantsColorectal cancer cases and matched controls were selected from among participants in theShanghai Women’s Health Study (SWHS), a prospective cohort of approximately 75,000women enrolled between 1997 and 2000 [14]. Of the 65,574 women in the SWHS cohortwho provided spot urine samples at enrollment, 347 were diagnosed with incident colorectalcancer through December 2005. One control was individually matched to each case based onage (±2 years), date of sample collection (within 1 month), menopausal status at samplecollection, time of sample collection (morning or afternoon), and time interval since last meal(within two hours). Four of the colorectal cancer cases were subsequently determined to havebeen misdiagnosed; these cases and the corresponding matched controls were excluded fromthe analyses of colorectal cancer risk, leaving 343 cases (colon cancers, N=205; rectalcancers, N=138) and matched controls.A total of 652 controls from the SWHS cohort were included in the analyses of determinantsof urinary 1-OHPG concentration (347 controls from the colorectal cancer study, and 305controls from a nested case–control study of gastric cancer for which samples were analyzedcontemporaneously at the same laboratory).Data and urine specimen collectionThe design and collection of data and specimens in the SWHS has been described [14].Briefly, women between 40–70 years of age were recruited from selected urban communitiesin Shanghai, China from 1997 to 2000. All subjects provided written informed consent, andthe study protocols were approved by the Institutional Review Boards of the National CancerInstitute, Vanderbilt University, and the Shanghai Cancer Institute. After obtaining informedconsent, subjects completed a self-administered questionnaire and an in-person interview.Information was collected on demographic characteristics, active and passive cigarettesmoking, diet and cooking practices, occupational history, and various other factors. Heightand weight were measured at the time of the in-person interview.Subjects were also asked to provide a spot urine sample, which was kept cold and processedwithin 6 hours for long-term storage. Another questionnaire was completed at the time ofsample collection to obtain additional information about diet, smoking, and medication usewithin the previous week and the 24-hour period prior to urine collection.Outcome ascertainmentColorectal cancer cases were identified through linkage with the Shanghai Cancer Registryand the Shanghai Vital Statistics Unit records, and in biennial follow-up visits withparticipants. Information on the date of cancer diagnosis was collected, and diagnoses wereverified by reviewing medical charts and diagnostic slides. Incident cases of colon and rectalcancers diagnosed through December 2005 were included in this analysis.

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Measurement of urinary 1-OHPG concentrationMeasurements of urinary concentration of 1-OHPG were performed using immunoaffinitychromatography and synchronous fluorescence spectroscopy [13]. Assays were performed inbatches of 20 including laboratory QC samples and blinded duplicate samples in each batch.The coefficient of variation (CV) for replicate measurements of urinary 1-OHPGconcentration across batches using aliquots from a single quality control pool was 10.6%. Inan analysis of blinded duplicate samples from 30 subjects, the Spearman rank correlationcoefficient for paired 1-OHPG measurements was 0.82.The assay limit of detection was 0.1 pmol/mL. For measurements below the limit of detection(7.6% and 7.3% of cases and controls, respectively), a value of 0.05 pmol/mL was assigned.Statistical analysisConditional logistic regression analyses were performed to estimate odds ratios (ORs) and95% confidence intervals (CIs) for risk of colorectal cancer in relation to creatinine-adjustedurinary 1-OHPG concentration, which was categorized as quartiles based on the distributionin controls. Statistical tests for trend were performed by modeling the within-categorymedians as a continuous parameter. We also evaluated colorectal cancer risk in relation tocreatinine-adjusted urinary 1-OHPG concentration as a continuous variable; as in previousstudies [15], a one unit change was defined as half the difference between the 25thand 75thpercentile measurements in controls. Analyses were performed with and without adjustmentfor the following covariates: age (years); education level; smoking status; aspirin use;estimated fruit, vegetable, and folate intake; measured body mass index (BMI); and leisuretime and occupational physical activity [in metabolic equivalent hours per week (MET-h/wk),as estimated in ref. [16]. Because intra-individual variability in 1-hydroxypyrene levels wasmodestly reduced in first morning void urine samples compared to 24-hour urine samplesafter adjustment for creatinine [17], we used creatinine-adjusted 1-OHPG concentrations inour main analyses. However, to evaluate whether correcting 1-OHPG measurements forcreatinine concentration had any effect on the resulting risk estimates, we performedsensitivity analyses using 1-OHPG concentrations without correction for creatinine, and withcreatinine concentration included as an independent variable in the statistical model, asrecommended by Barr et al. [18]. Several analyses were performed to assess whether theassociation differed by time from sample collection to case diagnosis, including an analysisrestricted to cases (and corresponding matched controls) diagnosed two or more years aftersample collection, and analyses stratified by duration of follow-up (below/above the mediantime of 3.8 years from sample collection to diagnosis, and above the 75thpercentile of follow-up time to diagnosis of 5.6 years). We also conducted analyses stratified by tumor location(colon vs. rectal) and menopausal status.As secondary analyses, we evaluated urinary 1-OHPG concentration as a dependent variablein relation to selected exposures that were suspected a priori to contribute to PAH exposure.These analyses were conducted among a combined set of control subjects (N=652).Measurements of 1-OHPG concentration were corrected for creatinine concentration, anddata were natural log-transformed to achieve a normal distribution. Results are reported as thegeometric mean (GM) and 95% CI within each exposure category. Self-reported exposuresfrom the baseline questionnaire that were evaluated included active and passive smokingstatus, cooking fuel type, cooking ventilation conditions, and food preparation methods. Wealso evaluated smoking and food preparation methods in the last week and last 24 hours prior

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consumption of fried dough products, and cooking with a coal stove), and no statisticallysignificant associations were observed (not shown).DiscussionIn this nested case–control study among participants in the SWHS, we did not find evidenceof an association between urinary concentration of 1-OHPG, an established marker of PAHexposure, and risk of colorectal cancer. There is considerable evidence from both animal andhuman studies that PAHs and PAH-containing materials are carcinogenic [reviewed in ref.[19]. Potential exposure to PAHs from employment as a gas furnace worker [4], smoking[10], and reported meat consumption [5-9] has been linked to colorectal cancer and colorectaladenoma. Each of these risk factors might involve exposure to various pyrolysis compounds,though PAHs are suspected to be among the agents responsible for the association withcolorectal cancer. In one case–control study, Sinha and colleagues [5] estimated dietaryintake of benzo(a)pyrene (a PAH metabolite) and its relation with colorectal adenoma risk.The investigators reported an odds ratio of 5.6 comparing subjects in the highest quintile ofestimated dietary benzo(a)pyrene intake relative to the lowest quintile, and a statisticallysignificant dose–response trend was observed [5]. However, in a more recent largeprospective study, estimated dietary intake of benzo(a)pyrene was not associated withcolorectal cancer risk [20].In the present study, we sought to further evaluate the hypothesis that PAH exposure isassociated with an increased risk of colorectal cancer using urinary 1-OHPG concentration asa quantitative biological measure of PAH exposure. Very few studies have evaluatedcolorectal cancer risk in relation to biological markers of PAH exposure. A hospital-basedcase–control study of colorectal adenoma among non-smoking U.S. residents by Gunter et al.[11] characterized PAH exposure based on the level of leukocyte PAH-DNA adducts, whichis considered to be a marker of relatively recent exposure [i.e., within the last week; ref. [21].In this case–control study, individuals in the highest quartile of leukocyte PAH-DNA adductlevels had a significantly greater risk of colorectal adenoma than individuals in the lowestquartile, and a statistically significant dose–response trend of increasing colorectal adenomarisk by quartile of PAH-DNA adduct level was observed. However, this study was limited bya relatively small number of cases of colorectal adenoma (N=82), and blood samples werecollected after diagnosis. More recently, Agudo et al. [12] conducted a case-cohort studyevaluating the relationship between aromatic DNA adduct levels in pre-diagnostic leukocytesand risk of colorectal cancer (154 cases). The investigators reported a statistically significantincreased risk of colorectal cancer with increasing levels of aromatic DNA adducts, andobserved a stronger association for colon cancer than for rectal cancer. In the present study,we were unable to confirm these findings using pre-diagnostic urinary 1-OHPGmeasurements in a larger sample of colorectal cancer cases and individually matchedcontrols.As a secondary analysis, we also evaluated potential determinants of urinary 1-OHPGconcentration among 652 control subjects from the SWHS cohort. This sample wascomprised of women ages 40 to 70 residing in Shanghai, China, only 4.6% of whom eversmoked cigarettes. In general, the levels of 1-OHPG in this population were considerablyhigher than what is typically observed among non-smokers in the United States, Europe andKorea, where median levels are approximately 0.3-0.4 pmol/mL [22,23]. The median(interquartile range) urinary concentration of 1-OHPG that we observed was 2.01 pmol/mL(0.95-4.09), which is comparable to levels observed in other populations in Linxian, China

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[24], northeastern Iran [25], and South Brazil [26], all of which are regions with well-characterized high levels of PAH exposure.Results of bivariate and multivariate analyses revealed that active and passive smoking,cooking with coal as a fuel source, eating meat or fish that was cooked until it was “entirelybrown”, and recent consumption of fried dough products influenced levels of 1-OHPG.However, only active smoking and recent consumption of fried dough products wereassociated with statistically significant increases in urinary 1-OHPG concentration afteradjustment for other covariates. Active cigarette smoking has been consistently associatedwith higher urinary levels of 1-OHPG in various populations [22,25-27]. It is reasonable toexpect that passive smoke exposure might also influence 1-OHPG levels. Our findingsregarding passive smoking were consistent with the results of a previous study ofdeterminants of urinary 1-OHPG concentration among children in South Korea, which foundthat the reported number of cigarettes smoked in the child’s home was associated withincreased 1-OHPG concentration [28].Diet, in particular consumption of charbroiled meats, has also been recognized as animportant determinant of 1-OHPG concentration [1,23]. Lee et al. [28] reported thatconsumption of grilled fish, but not grilled meats, was associated with children’s’ levels of 1-OHPG. We found that the “doneness” of fried or baked meat or fish was associated with 1-OHPG concentration, though results were not statistically significant. Consumption of frieddough products – such as yóutiáo, a deep-fried twisted dough stick that is a popular breakfastfood often purchased from street vendors or in snack shops – could potentially be animportant dietary source of PAH exposure. High levels of benzo(a)pyrene have been detectedin cooking oil fumes from shops selling yóutiáo [29], and it is possible that individuals mightbe exposed via ingestion of dough that has been fried in cooking oil containing PAHs. Thisfinding suggests that consumption of fried dough foods such as yóutiáo should be consideredin future studies that assess dietary PAH exposure in Chinese populations. Though very fewsubjects never ate stir-fried meats (N=20), we observed higher 1-OHPG levels in this group.This finding was unexpected, and no such association between 1-OHPG levels and recentconsumption of stir-fried or roasted meats was observed. However, it is possible that thisassociation may reflect other differences in diet, and further investigation of dietary PAHexposure among these individuals may reveal other potential sources of exposure.Strengths and limitationsThe large sample size, prospective collection of urine samples, and availability of data onpotential confounding factors were strengths of this study. Because samples were collectedprospectively and results were similar when cases diagnosed within two years of samplecollection were excluded, it is unlikely that early disease processes would have affected 1-OHPG measurements. The availability of prospectively collected data on covariatesminimized potential recall bias and allowed us to control for potential confounding factorssuch as age, BMI, physical activity, and fruit and vegetable intake in our analyses.There were also several limitations in this study. Urine was collected as a spot sample forpractical reasons, though the interpretation of urinary creatinine levels used for the correctionto ≥24 hour urine output may be problematic. To address this issue, we performed sensitivityanalyses without correction for creatinine and with adjustment for creatinine concentration asa covariate in the statistical model; results were similar to what was observed in the mainanalyses.

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As with most prospective investigations involving a single pre-diagnostic specimen, it ispossible that urinary levels of 1-OHPG may not have reflected long-term PAH exposurestatus or exposure levels during the etiologically relevant period. In our analyses amongcontrols, we identified associations between several reported sources of exposure from thebaseline and sample collection questionnaires and urinary concentration of 1-OHPG,suggesting that 1-OHPG levels may be a reasonable reflection of both usual and recentexposure to PAHs. It should be noted that when we evaluated colorectal cancer risk inrelation to these sources of PAH exposure, our findings were similarly null. The median timefrom urine sample collection until diagnosis of colorectal cancer in this study was 3.8 years;continued follow-up of the cohort may be useful to assess relations between long-term PAHexposure and colorectal cancer risk.Most previous studies of determinants of 1-OHPG concentration have been based onrelatively small sample sizes, and the inclusion of a large number of healthy subjects (N =652) was a strength of the present study. However, despite the large sample size, some of thereported exposures were relatively uncommon in this population. Also, there was somewhatless heterogeneity of 1-OHPG levels relative to other highly exposed populations [25,26],which may have limited our ability to detect associations with colorectal cancer and toevaluate determinants of PAH exposure. We were unable to assess ambient exposure toPAHs in this study, an important determinant of exposure in previous studies [30] that couldpossibly explain the high and relatively homogeneous levels of 1-OHPG in our studypopulation.ConclusionsWe did not find evidence of an association between urinary 1-OHPG levels and colorectalcancer risk in this nested case–control study. Future studies with greater duration of follow-up and characterization of PAH exposure over a longer time period are needed to betterunderstand the role of PAHs in the etiology of colorectal cancer.Abbreviations1-OHPG, 1-hydroxypyrene glucuronide; BMI, Body mass index; CI, Confidence interval;CV, Coefficient of variation; GM, Geometric mean; MET-h/wk, Metabolic equivalent hoursper week; OR, Odds ratio; PAH, Polycyclic aromatic hydrocarbon; QC, Quality control;SWHS, Shanghai Women’s Health StudyCompeting interestsThe authors declare that they have no competing financial interests.Authors’ contributionsJNH conducted the data analysis and drafted the manuscript; LML advised on the statisticalanalyses and helped draft the manuscript; PTS conducted the assays measuring urinary 1-OHPG levels and provided important intellectual content; XOS, GY, BTJ, HLL, NR, YTG,and WZ oversaw subject recruitment and data collection for the cohort study and providedintellectual input into preparation of the manuscript; FK and WHC conceptualized the study,